Recent evidence suggests that the human transcriptome is not only significantly larger than previously recognized but also it is primarily composed of functional RNA transcripts which are not translated into proteins known as non-coding RNAs (ENCODE Project Consortium, Nature 2007, v.447, p.799-816). Among the more intensely studied class of endogenously expressed non-coding RNAs are the micro-RNAs (miRNAs). Mature miRNAs are relatively small (21-23 nucleotides) RNA duplexes that act as translational repressors of protein expression. The guide strand of a miRNA unites with Argonaute family proteins (Ago) to form RNA-Induced Silencing Complexes (RISC) in the cell. These sequence-specific ribonucleoprotein complexes bind target mRNAs typically in the 3′UTR and can subsequently silence gene expression either through directed mRNA degradation or by simply sequestering the target mRNA in an ineffectual form (Lee et al., Cell 1993, v.75, p.843-854; Bartel, Cell 2009 v.136, p. 215-233). It has been demonstrated that miRNA based regulation plays a significant role in routine cellular processes including metabolism (Esau et al, Cell Met. 2006, v.3, p 87-98), development (Carthew et al., Cell 2009, v.137, p.273-282), and even apoptosis (Cheng et al, Nucl. Acids Res. 2005, v.33, p1290-1297). Further research has revealed that miRNAs play critical roles in diverse disease processes such as hepatitis C (Jopling et al., Science 2005, v.309, p.1577-1581), diabetes (Poy et al., Nature 2004, v.432, p.226-230), and most notably multiple cancer types (Hammond, Can. Chemo. Pharma. 2006 v.58, s63-s68; Calin et al., Cancer Res. 2006, v.66, p.7390-7394) including leukemia (Calin et al., PNAS 2002, v.101, p.2999-3004) and glioma (Corsten et al., Cancer Res. 2007, v.67, p.8994-9000). In addition, miRNA discovery has far surpassed miRNA phenotypic identification thus creating a “validation gap” for miRNA function (Griffiths-Jones et al., Nucl. Acids Res. 2006, v.34, p.D141-D144). Over one thousand miRNAs have now been identified in animals, but only a few individual miRNAs have been linked to specific functions.
Given the range and degree of effects that miRNAs have on cellular processes and that a single miRNA can modulate multiple gene products (Selbach et al., Nature 2008, v.455, p.58-63), miRNAs have become attractive targets both for loss of function studies in vitro (to study miRNA function and mechanism) and potential therapeutic applications in vivo. To this end, research groups both public and private have sought to develop highly potent miRNA inhibitors (antisense oligos which bind to complementary miRNAs and selectively block silencing in cell extracts, in cultured cells, and in vivo) by utilizing one of three basic approaches: (i) antisense (AS)-based oligonucleotides that employ chemically modified sugars, bases, phosphate backbones, and/or terminal conjugates (Krutzfeldt et al., Nucl. Acids Res. 2007 v.35, p.2885-2892, Horwich et al., Nat. Protocols, 2008 v.3, p.1537-1549, Davis et al., Nucl. Acids Res. 2006, v.34, p.2294-2304); (ii) long (>34 nucleotides) oligonucleotides wherein the reverse complement (AS) strand to the miRNA is flanked on both the 3′ and 5′ end with 12-16 nucleotides which are intended to form hairpin loops (Vermeulen et al., RNA 2007, v.13, p.723-730); and (iii) Tandemic antisense RNA produced from DNA vectors with multiple miRNA binding sites per unit which behave as decoy targets for endogenous miRNAs (“miRNA sponges”, Ebert et al, Nature Methods 2007, v.4, p.721-726). Further, 2′ O-Me oligonucleotides have been produced to be resistant to cleavage by RISC and other cellular ribonucleases (Hutvágner, et al., PloS Biol. 2004, v.2, p. E98; Meister, et al., RNA 2004, v.10, p.544-550). Oligonucleotides have also been made that combine 2′-deoxy and locked nucleic acid (LNA) nucleotides (Lecellier et al., Science 2005, v.308, p.557-560). Oligonucleotides have been made that contain all 2′-O-methoxyethyl nucleotides and oligonucleotides incorporating pyrimidines bearing 2′-O-fluoromodifications (Essau et al., Cell Metab. 2006, v. 3, p.87-89; Davis et al, Nucleic Acids Res. 2006, v. 34, p.2294-2304). Further, nuclease resistant phosphorothioate backbone linkages in combination with ribose modifications have also been employed in cultured cells and in vivo in mice and non-human primates (Essau et al., Cell Metab. 2006, v.3, p.87-89; Elmén et al., Nature, 2008, v. 452, p. 896-899).
Life Technologies currently offers miRNA inhibitors with proprietary modifications (AntiMiRs), Exiqon offers LNA-modified short antisense inhibitors, and Dharmacon/ThermoFisher offers 2′-OMe antisense inhibitors with hairpin structure motifs at both the 3′ and 5′ ends (>55 nt). The invention discussed herein offers several novel and unique designs for miRNA inhibitors that enable superior miRNA inhibition.